Abstract The PD-1:PD-L1 pathway plays multifaceted roles in controlling the balance between pathogenic and protective immune responses in T cell tolerance and autoimmunity. During the current funding period, our studies have revealed critical roles for the PD-1:PD-L1 pathway in regulating T cell activation, tolerance and immune-mediated tissue damage. We have found that PD-L1 not only inhibits the activation and function of pathogenic effector T cells, but also promotes the development and sustains the function of induced regulatory T cells. In addition, we have identified a novel role for PD-L1 on non-hematopoietic cells in protecting target organs from immune-mediated tissue injury. However, we lack a mechanistic understanding of the function of PD-L1 on different cell types. The great interest in PD-L1 and PD-1 as therapeutic targets gives impetus to further investigation of how these important immunoregulatory molecules regulate autoreactive T cells. The overall goal of this project is to elucidate mechanisms by which PD-L1 controls T cell tolerance, prevents autoimmunity, and limits immune-mediated inflammation locally within tissues. Our published and preliminary data lead us to hypothesize that PD-L1 on specific types of hematopoietic and non-hematopoietic cells will regulate T cell activation, tolerance and autoimmunity-related pathology by distinct mechanisms. We will use our newly generated PD-L1 conditional knockout mice crossed to cell type-specific Cre lines to test this hypothesis in the following Specific Aims. Aim 1: To test the hypothesis that there are distinct roles for PD-L1 on specific hematopoietic or non-hematopoietic cell types in regulating the activation, differentiation, and function of potentially pathogenic self-reactive T cells. We will analyze the relative functions of PD-L1 on specific hematopoietic and non-hematopoietic cell types in controlling the activation and differentiation of nave myelin-reactive CD4 T cells and the functions of differentiated myelin-reactive effector cells using the myelin oligodendrocyte (MOG) model of experimental autoimmune encephalomyelitis (EAE). 2) To test the hypothesis that there are distinct roles for PD-L1 on specific cell types in controlling the generation and function of regulatory T cells. We will analyze the relative functions of PD-L1 in controlling natural regulatory T cells (nTreg) and induced regulatory T cells (iTreg). We will use the MOG-induced EAE model to study how PD-L1 regulates nTreg expansion, function and plasticity. We will study how PD-L1 on specific cell types promotes iTreg generation, function, and plasticity using a colitis model, where iTreg are best defined. These studies should further our understanding of mechanisms that control tolerance and autoimmunity, and provide insight into how to modulate PD-L1 and PD-1 therapeutically to control autoimmune diseases, chronic infections and cancer.